silencing onion lachrymatory factor synthase causes a ... · silencing onion lachrymatory factor...

Silencing Onion Lachrymatory Factor Synthase Causesa Significant Change in the Sulfur SecondaryMetabolite Profile1[W][OA]

Colin C. Eady*, Takahiro Kamoi, Masahiro Kato, Noel G. Porter, Sheree Davis, Martin Shaw,Akiko Kamoi, and Shinsuke Imai

National Centre for Advanced Bio-Protection Technologies, Lincoln University, Christchurch 7647,New Zealand (C.C.E., T.K.); New Zealand Institute for Crop and Food Research Limited,Christchurch 8140, New Zealand (C.C.E., N.G.P., S.D., M.S.); and House Foods Corporation,Yotsukaido, Chiba 284–0033, Japan (T.K., M.K., A.K., S.I.)

Through a single genetic transformation in onion (Allium cepa), a crop recalcitrant to genetic transformation, we suppressed thelachrymatory factor synthase gene using RNA interference silencing in six plants. This reduced lachrymatory synthase activityby up to 1,544-fold, so that when wounded the onions produced significantly reduced levels of tear-inducing lachrymatoryfactor. We then confirmed, through a novel colorimetric assay, that this silencing had shifted the trans-S-1-propenyl-L-cysteinesulfoxide breakdown pathway so that more 1-propenyl sulfenic acid was converted into di-1-propenyl thiosulfinate. Aconsequence of this raised thiosulfinate level was a marked increase in the downstream production of a nonenzymaticallyproduced zwiebelane isomer and other volatile sulfur compounds, di-1-propenyl disulfide and 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene, which had previously been reported in trace amounts or had not been detected in onion. The consequencesof this dramatic simultaneous down- and up-regulation of secondary sulfur products on the health and flavor attributes of theonion are discussed.

Allium species synthesize a unique set of secondarysulfur metabolites derived from Cys. Most notable arethe S-alk(en)yl-L-Cys sulfoxides, including S-2-propenyl-L-cysteine sulfoxide (alliin; 2-PRENCSO) and trans-S-1-propenyl-L-cysteine sulfoxide (isoalliin; 1-PRENCSO;Rose et al., 2005). When the tissues of any Alliumspecies are disrupted, these amino acid derivatives arecleaved by the enzyme alliinase (EC 4.4.1.4) into theircorresponding sulfenic acids, and volatile sulfur com-pounds are produced that give the characteristic flavorand bioactivity of the species. In garlic (Allium sat-ivum), 2-PRENCSO is the major sulfoxide (Fritsch andKeusgen, 2006), and this produces di-2-propenyl thio-sulfinate (allicin) upon tissue disruption. The decom-position product of allicin, di-2-propenyl disulfide, isthe dominant volatile component liberated (Blocket al., 1992a, 1992b; Rose et al., 2005). In onion (Alliumcepa), 1-PRENCSO is the major sulfoxide (Fritsch and

Keusgen, 2006). This would be predicted to producedi-1-propenyl thiosulfinate and di-1-propenyl disul-fide. However, di-1-propenyl thiosulfinate has neverbeen reported in onion, and di-1- propenyl disulfidehas only tentatively been reported in trace amounts(Boelens et al., 1971). Instead, propanthial S-oxide(lachrymatory factor [LF]), 1-propenyl methane thio-sulfinate, and di-propyl disulfide are dominant (Blocket al., 1992a, 1992b; Rose et al., 2005). LF is a criticalpractical point of difference between onion and garlic.It is the chemical responsible for inducing tearing inonion, an undesirable irritant, and it is hypothesizedthat LF production causes the absence of otherwisepredicted sulfur volatiles (Randle and Lancaster,2002), analogues of which in garlic are known fortheir health attributes (Griffiths et al., 2002).

Imai et al. (2002) discovered that the conversion of1-propenyl sulfenic acid to LF is mediated by an enzymethey named lachrymatory factor synthase (LFS; Fig. 1).LFS acts specifically upon 1-propenyl sulfenic acid,following the action of alliinase on 1-PRENCSO toproduce LF. The specificity of this end pathway reac-tion suggests that the production of LF could bereduced by genetic manipulation of the LFS transcriptusing RNA interference (RNAi) silencing. In the ab-sence of LFS, the unstable 1-propenyl sulfenic acidwould undergo spontaneous self-condensation to di-1-propenyl thiosulfinate (Imai et al., 2002). This raisedthiosulfinate level would then be available for conver-sion, via the nonenzymatic part of the pathway (Fig. 1),into a cascade of predicted secondary compounds that

1 This work was supported by the New Zealand Foundation forResearch, Science, and Technology and by House Foods Corporationof Japan.

* Corresponding author; e-mail [email protected] author responsible for distribution of materials integral to the

findings presented in this article in accordance with the policydescribed in the Instructions for Authors (www.plantphysiol.org) is:Colin C. Eady ([email protected]).

[W] The online version of this article contains Web-only data.[OA] Open Access articles can be viewed online without a sub-

scription.www.plantphysiol.org/cgi/doi/10.1104/pp.108.123273

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have not been detected previously in onion or haveonly been reported in trace amounts. These com-pounds are responsible for the unique sensory, odor,and flavor notes (Block, 1992) as well for as the health-promoting attributes of the edible Allium species (e.g.antiinflammatory, antiplatelet aggregation, anticancer,lipid-lowering; Morimitsu et al., 1992; Block et al.,1996b; Griffiths et al., 2002; Randle and Lancaster,2002; Lanzotti, 2006).

Onion is a very difficult biological system to workwith; it has a large genome, is heterozygous, has a slowgeneration time, and has a poor capacity for manipu-lation (Eady, 2002; Eady and Hunger, 2008). However,Allium is a genus with a unique, complex, and impor-tant secondary sulfur pathway that has no modelcounterpart. Current ‘‘tearless’’ onion cultivars (e.g.‘Vidalia’) are achieved through deficient uptake andpartitioning of sulfur and/or growth in sulfur-deficientsoils, but in so doing they accumulate fewer secondarysulfur compounds in the bulb (Randle and Lancaster,2002), reducing their sensory and health qualitiescompared with more pungent high-sulfur cultivars.In this research, we set out to genetically manipulatethe sulfur secondary metabolite pathway of onionusing RNAi. Unlike previous research aiming to over-regulate or underregulate a particular enzyme withina secondary pathway to either increase consumer-desirable compounds (Davuluri et al., 2005) or removedeleterious ones (Capell and Christou, 2004; Sunilkumaret al., 2006), we aimed to achieve both. By reducingLFS and stopping the conversion of 1-propenyl sul-fenic acid to the undesirable LF, we tested the hypoth-esis that this would allow 1-propenyl sulfenic acid to

be available for spontaneous conversion into thiosul-finate and thiosulfinate-derived sulfur compounds,analogues of which are renowned for their desirablesensory and health-promoting attributes.

RESULTS

Three onion cultivars were studied: a mild hybrid(H) mid-daylength fresh onion (‘Enterprise’), a pun-gent open-pollinated (O) fresh onion (‘PukekoheLongKeeper’), and a pungent dehydration (D) mid-daylength onion (Sensient Dehydrated Flavors).Eleven plants were evaluated, three nontransgenicplants (HN, ON, and DN) and eight transgenic plants(H1, H2, H3, O1, O2, O3, D1, and D2), from the hybrid,open-pollinated, and dehydration cultivars as indi-cated. These were the transformants recovered fromapproximately 15,000 immature embryos used in 16transformation experiments (approximately 0.05% trans-formation frequency).

Plant Selection and Regeneration

Under selection and regeneration, the transformedtissue behaved in a similar manner to that observed inprevious onion transformations (Eady et al., 2000,2002). Transgenic shoot cultures rooted well in me-dium containing geneticin, except for plant O3, whichhad to be rescued onto nonselective medium. Alltransgenic plants grew and formed morphologicallysimilar plants and bulbs to their nontransgenic coun-terparts (Fig. 2). Seed set and F1 progeny had beenobtained from two lines by selfing or crossing ontonontransgenic counterparts.

T-DNA Integration and Integrity

Southern-blot analysis of onion plants, using a gfpgene probe (Eady et al., 2000), revealed that plants H1and D1 contained two copies of the T-DNA construct atdifferent loci and that plant O1 contained a multipleinsert at a single locus. The remaining five plants, H2,H3, O2, O3, and D2, contained single-copy inserts (Fig.3, top) integrated at different locations from each other,confirming the nonclonal nature of the transgenicevents. PCR data (Table I) indicated that the T-DNAcassette was not complete in all plants evaluated. Inplant O3, the nptII gene sequence could not be detected.Initial identification of this transgenic event by GFPexpression and rescue to nonselective medium resultedin the maintenance of this plant. In plant H2, the 5#region of the lfsRNAi cauliflower mosaic virus (CaMV)35S promoter sequence was truncated. However, thisdid not compromise transcription or small interferingRNA (siRNA) production (Fig. 3, center; Table I).

siRNA Production

Detection of lfsRNAi transcript by reverse transcrip-tion (RT)-PCR was used to indicate functionality of the

Figure 1. The main sulfur pathway following tissue disruption in onion.

Silencing Lachrymatory Factor Synthase in Onion

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transgene. All transgenic plants except O1 producedlfsRNAi transcript (Table I). Such observation of trans-gene inactivation due to multiple-copy inserts at asingle locus is common (Muskens et al., 2000; Tanget al., 2007). Detection of lfs siRNA using a lfs probe(Fig. 3, center) showed that six plants (H1, H2, H3, O2,O3, and D2) produced siRNA fragments correspond-ing to the LFS gene sequence. Interestingly, plant D1,which produced lfsRNAi transcript, failed to producelfs siRNA at detectable levels. We are unaware of otherresearch that has used RT-PCR to detect hairpin tran-scripts and suggest that it is a valuable tool to differ-entiate reasons for hairpins failing to silence. In thiscase, we can assert that the cause was not transcrip-tional inactivation.

LFS Levels

lfs transcript levels (Fig. 3, bottom) were comparedin cDNA samples from transgenic and nontransgenicplants by quantitative RT-PCR. Low levels of tran-

script corresponded well with the presence of lfssiRNA fragments.

No LFS protein could be detected in plants thatproduced lfs siRNA. Plants O1 and D1, with no ob-servable lfs siRNA, had LFS protein levels that fell wellwithin the range of their respective control nontrans-genic plants (Fig. 4, top).

Assays of LFS activity in both leaf and bulb mea-sured by in vitro generation of LF demonstrated thatplants with no detectable LFS, as measured by western-blot analysis, also had significantly reduced LFS activity(Fig. 4, middle). This activity in plants H1, H2, and H3was reduced by between 21- and 103-fold in leaf tissueand by between 18- and 1,168-fold in bulb tissue.Activity in plants O2 and O3 was reduced by between38- and 70-fold in leaf tissue and by between 1,515- and1,544-fold in bulb tissue. Activity in plant D2 wasreduced by 396-fold in leaf and by 501-fold in bulbtissue. The more pronounced reduction observed inbulb tissue over leaf tissue suggests that LFS is probablya relatively major protein within aestivating storage

Figure 2. A to C, Ex-flasked intermediate daylengthopen-pollinated (O lines; A), hybrid (H lines; B), anddehydration (D lines; C) onions transformed with thelfsRNAi construct. D, LFS-silenced bulbs.

Table I. Summary of molecular and biochemical data for transgenic onion plants

AnalysisPlants

H1 H2 H3 O1 O2 O3 D1 D2

PCR IntactT-DNA

CaMV 35Sdeleted

IntactT-DNA

IntactT-DNA

IntactT-DNA

nptIIdeleted

IntactT-DNA

IntactT-DNA

Southernblot

Two atdifferentloci

Singleinsert

Singleinsert

Multipleinserts atone locus

Singleinsert

Singleinsert

Two atdifferentloci

Singleinsert

lfsRNAitranscripts

Present Present Present Absent Present Present Present Present

siRNAs Present Present Present Absent Present Present Absent Presentlfs transcripts Reduced Reduced Reduced Normal Reduced Reduced Normal ReducedLFS protein Reduced Reduced Reduced Normal Reduced Reduced Normal ReducedLFS activity Reduced Reduced Reduced Normal Reduced Reduced Normal Reduced

Eady et al.

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bulb tissue compared with leaf material. Plants D1 andO1 failed to produce the LFS silencing signal or toreduce LFS activity.

Precursor 1-PRENCSO Levels and Alliinase Activity

Biochemical analysis showed that the 1-PRENCSOlevels in the transgenic and control plants were be-tween 4 and 13 mg g21 dry weight. Alliinase activitywas between 15.8 and 42.4 nkat mg21 protein. Thesesubstrate and enzyme levels are within the normalphysiological range reported for onion (Kitamuraet al., 1997; Kopsel and Randle, 1999). This suggeststhat in our transgenic onions, silencing lfs transcriptsdid not affect alliinase activity or 1-PRENCSO levels.

Phenotype Analysis of Secondary Sulfur Chemistry

In order to identify all of the possible changes toonion secondary sulfur metabolism, three establishedtechniques were used: gas chromatography (GC) withflame photometric detection, solid-phase microextrac-tion (SPME) GC-mass spectrometry (MS), and solventextraction GC-MS. In addition, to detect the previouslyundetected in onion di-1-propenyl thiosulfinate, anovel colorimetric (‘‘pinking’’) assay was developedand used.

Volatile Sulfur Compounds

GC-flame photometric detection analysis of LFlevels from freshly crushed leaf material demonstratedthat H1, H2, and H3 were reduced by 13.5-, 35.5-, and30-fold, respectively, compared with HN, that O2 andO3 were reduced by 30- and 67-fold compared withON, and that D2 was reduced by 36-fold comparedwith DN. In bulb material, LF was reduced by 10.2-and 28.2-fold for H1 and H3 compared with HN (H2

Figure 3. Molecular analysis. Top, Southern analysis of lfsRNAi trans-genic onion plants probed with gfp probe. Center, RNAi analysis ofsmall RNA probed with lfs probe. Bottom, Quantitative RT-PCR mea-surement of lfs transcript compared with nontransgenic (NT). HN, ON,and DN indicate nontransgenic hybrid, open-pollinated, and dehydra-tion control, respectively (stars); H1, H2, H3, O1, O2, O3, D1, and D2represent specific transgenic hybrid, open-pollinated, and dehydrationonion plants (circles). Far right lanes show one-, five-, and 10-copy con-trol, respectively (on Southern-blot gel) and LFS sequence control (onRNAi gel). The 95% confidence limit is calculated from an ANOVA oflogged data using the within-bulb variation. Data were log-transformed toadjust for variance heterogeneity between lines.

Figure 4. Biochemical analysis. Top, Western-blot analysis of LFS.Middle, In vitro LF peak area (LPA). Bottom, In vivo LF measurements.HN, ON, and DN indicate nontransgenic hybrid, open-pollinated, anddehydration control, respectively; H1, H2, H3, O1, O2, O3, D1, andD2 represent specific transgenic hybrid, open-pollinated, and dehy-dration onion plants. The 95% confidence limit is calculated byanalysis of log-transformed data of within-bulb and within-leaf varia-tion to account for the variance difference between the low and high LFvalues. ND, Not detected.





was not measured, as the bulb was infected), by 6.4-and 28-fold for O2 and O3 compared with ON, and by12.8-fold for D2 compared with DN (Fig. 4, bottom).Analysis of F1 progeny from line D2 has demonstratedthat the reduced LF level is inherited along with GFPexpression (data not shown).

Other sulfur volatile components given off by dam-aged onion tissue were detected by SPME-GC-MSanalysis of head space, as proposed by Arnault et al.(2000). Healthy cut leaf blade material was analyzedusing a protocol similar to that described by Hori(2007). The identification of the volatile compoundswas based on comparisons of mass spectral data (Sup-plemental Fig. S1) with published work (Hiramitsu,1989; Sinha et al., 1992; Block et al., 1996a). Thisanalysis (Fig. 5) revealed that the LF-reduced plantsproduced a significantly decreased di-propyl disul-fide peak and a much increased 1-propenyl propyldisulfide peak compared with control plants. In addi-tion, the LF-reduced plants produced five peaks notdetected in the control onion: three peaks representingdi-1-propenyl disulfide isomers and two peaks repre-senting 2-mercapto-3,4-dimethyl-2,3-dihydrothiophene

isomers. The disulfides have only previously beenreported in trace amounts in onion (Boelens et al.,1971). The dihydrothiophenes have only previouslybeen reported in trace amounts in Welsh onion (Kuoand Ho, 1992a, 1992b) but have never been detected inonion.

Subjective sensory evaluation of the reduced-LFplants by the authors supported the GC analysis.Onion leaf and bulb samples (0.5 cm2) crushed be-tween fingernails and placed touching the eyelashescaused a stinging and tearing sensation with controltissue but no tearing or stinging when reduced-LFmaterial was used. The same material when placednext to the nose revealed an aroma in the reduced-LFplants that was less pungent and sweeter than thatgiven off by the nontransgenic counterparts.

Thiosulfinate Detection and Abundance

A key prediction was that the reduction of LFS levelswould leave more 1-PRENCSO-derived 1-propenylsulfenic acid available for thiosulfinate production.However, no simple method of specifically detecting

Figure 5. GC analysis of solid-phase microextractionsulfur components from the head space of vialscontaining cut onion leaf material. Peak 1, Dipropyldisulfide; peak 2, 1-propenyl propyl disulfide; peak 3,di-1-propenyl disulfide isomer 1; peak 4, di-1-propenyldisulfide isomer 2; peak 5, di-1-propenyl disulfide isomer3; peak 6, syn-2-mercapto-3,4-dimethyl-2,3-dihydro-thiophene; peak 7, anti-2-mercapto-3,4-dimethyl-2,3-dihydrothiophene.

Eady et al.




di-1-propenyl thiosulfinate was available. Block(1991) synthesized the thiosulfinate by oxidation ofdi-1-propenyl disulfide and demonstrated by low-temperature NMR analysis at 215�C that it rapidlychanged form to become a zwiebelane; thus, its ther-mal instability ruled out the use of standard GCtechniques for analysis. Recently, observations byImai et al. (2006a, 2006b) demonstrated that 1-propenyl-containing thiosulfinates produce a pink pigment (ofunknown structure) when mixed with Gly and form-aldehyde. We exploited this reaction to develop asimple colorimetric assay (pinking assay) to detect1-PRENCSO-derived thiosulfinate (Supplemental Fig.S2). The specificity of detection was confirmed by invitro analysis of 1-PRENCSO- or alliin (2-PRENCSO)-derived products incubated in the presence of garlicalliinase (Fig. 6). The 1-PRENCSO-derived productcorrelated with a pink color, while the alliin-derivedproduct produced no pink color. Pinking analysis ofthe reduced-LF plants confirmed increased levels of1-propenyl-containing thiosulfinates. Furthermore, ad-dition of recombinant LFS to the extract lowered thepinking level and confirmed that the absence of LFSwas the cause of pinking (Fig. 7). In vitro analysisindicated that not all 1-PRENCSO-derived thiosulfi-nates are captured in the color reaction and that LFS isa strong competitor for 1-propenyl sulfenic acid. In invitro assays, one to two times normal physiologicallevels of LFS (using recombinant LFS) converted morethan 85% of 1-PRENCSO to LF (data not shown).

Nonenzymatic Thiosulfinate-Derived

Reaction Products

The thiosulfinates are known to convert spontane-ously to a number of other sulfur compounds (Griffithset al., 2002), and solvent extraction GC-MS analysis ofcrushed reduced-LF plants was undertaken in aneffort to identify differences from nontransgenic on-ions. The total ion chromatogram produced by GC-MSanalysis of incubated reduced-LF extract from plantsH3, O3, and D2 compared with their nontransgeniccounterparts detected a large peak difference in thetransgenic extracts (Fig. 8), which was confirmed bythe mass spectra fingerprint (Supplemental Fig. S1) tobe a zwiebelane isomer. Zwiebelane isomers were first

reported by Arnault et al. (2000), although they did notidentify the chemical structure. Our analysis demon-strated that at least one of the proposed downstreamsulfur compounds was elevated in the reduced-LFonions.

DISCUSSION

This report is the first demonstration, to our knowl-edge, of gene silencing in an Allium species. It alsoshows that the manipulation of plant secondary me-tabolite pathways can result in dramatic simultaneousdown- and up-regulation of products within thatpathway and the production of novel products upontissue disruption.

Earlier work using antisense technology to silencethe alliinase gene in onion produced some transgenicplants, but conclusive proof of silencing was notobtained (Eady et al., 2004). In this work, we success-fully decreased endogenous LFS production through asingle simple transformation event. This unambigu-ously lowered LFS activity in the plant, and theconsequence of this was a significant reduction in theproduction of the irritant LF. This caused a large shiftin the 1-PRENCSO breakdown pathway such that muchmore 1-propenyl sulfenic acid was available for conver-sion into thiosulfinates. Raised levels of 1-propenyl-containing thiosulfinate (detected using the novelpinking assay) predictably increased levels of thenonenzymatically produced downstream product,a zwiebelane isomer and di-1-propenyl disulfides.

Figure 6. Pinking assay results showing specificity for 1-PRENCSOover alliin (2-PRENCSO).

Figure 7. Pinking assay results showing thiosulfinate abundance intransgenic and nontransgenic plants. Thiosulfinate abundance shownin nontransgenic (HN, ON, and DN) and transgenic (H3, O3, and D2)onion extracts with (squares) and without (circles) the addition ofrecombinant LFS. Images below show pinking color for the respectiveplants (the left Eppendorf is without recombinant LFS, the right is withrecombinant LFS). The 95% confidence limit is calculated from anANOVA using the within-bulb variation.





However, much more surprising and unpredictedwas the production of 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes.

Silencing the lfs transcript has confirmed the hy-pothesis that LF production prevents the spontaneousproduction, from 1-propenyl sulfenic acid, of 1-propenyl-containing thiosulfinates in crushed onion. This demon-strates the specificity and affinity of LFS for 1-propenylsulfenic acid and would indicate that alliinase and LFSmust work in close proximity, as negligible amountsescape conversion into LF.

The development of the pinking assay reported hereoriginated from in-depth studies of the cause of ‘‘offcolors’’ in processed onion and garlic products (Imaiet al., 2006a, 2006b). It provides a unique and simplemethod for detecting the 1-PRENCSO-derived thio-sulfinate. Unlike the N-ethylmaleimide method of Leeand Parkin (1998), which detects all thiosulfinates, thispinking method is specific to 1-propenyl thiosulfinate.The development of a high-throughput protocol for

this assay will provide Allium breeders and processorswith a simple tool to quantify this important precursor.

Previous research in Allium species (Boelens et al.,1971; Block and Zhao, 1990; Arnault et al., 2000) madeit possible to predict the nonenzymatic breakdown of1-propenyl-containing thiosulfinate (Fig. 9). Much ofwhat was predicted was indeed confirmed by ourresearch, with a zwiebelane isomer and 1-propenylpropyl disulfide and the di-1-propenyl disulfides be-ing detected in much greater abundance in volatilesfrom the reduced-LF plants. Arnault et al. (2000, 2004)reported that thiosulfinates and zwiebelanes are con-verted into disulfides in SPME-GC-MS analysis be-cause of its thermally severe condition compared withsolvent extraction GC-MS. The increase of 1-propenyl-containing disulfides in the SPME-GC-MS analysiscorresponded to increased absorbance in the pinkingassay of the reduced-LF plant extracts, indicatingthat the disulfides are breakdown products from the1-propenyl thiosulfinate. The decrease in dipropyl di-

Figure 8. Total ion chromatograms of incubated on-ion extract. Chromatograms for H3 and HN, O3 andON, and D2 and DN show the control benzyl alcoholand the zwiebelane isomer.

Eady et al.




sulfide level is most likely a result of the greater abilityof the propyl sulfenic acid to react with the propenylform or a result of the decrease of LF (Block, 1991).

The presence of 2-mercapto-3,4-dimethyl-2,3-dihydrothiophenes was not predicted. The dihydro-thiophenes were first reported to be formed by heatingdi-1-propenyl disulfide (Block and Zhao, 1990). Thus,the dihydrothiophenes detected in reduced-LF plantsare likely to be formed from di-1-propenyl disulfide inthermally severe SPME-GC-MS analysis. The resultspresented here indicate that the di-1-propenyl thio-sulfinate and its corresponding di-1-propenyl disul-fide are thermally very unstable and as such difficultto assess quantitatively, despite the use of standardGC-MS protocols (Arnault et al., 2000, 2004; Hori,2007). Thus, while the disulfides and dihydrothio-phenes may not be present in raw reduced-LF onion,they are likely to be produced in a cooked reduced-LF

onion. Further research will clarify the levels of thesereaction products in onion.

The impact on flavor of the lower LF levels com-bined with the raised levels of thiosulfinate-derivedsulfur compounds in the reduced-LF onion can bepredicted from the existing literature. LF is the causeof the unpleasant pungent aroma associated with cutonions and the cause of heat and pungency flavornotes (Randle, 1997). Disulfide and dihydrothiophenecompounds are associated with the sweeter aroma ofcooked or fried onions (Lancaster and Boland, 1990;Albanese and Fontijne, 2002). Trans- and cis-zwiebelanesare associated with sweet raw onion sulfur tastes andsweet brown saute flavor notes, respectively (Blocket al., 1996b). Initial olfactory assessment by the au-thors (smelling the crushed onion samples) indicatedthat the pungent aroma was absent in the reduced-LFplants and that the sweeter aroma of cooked onionswas present. The literature suggests that the reduced-LF onions will have low heat and pungency combinedwith enhanced sweet raw onion and saute flavor notes.Formalized taste evaluation of the transgenic plantsrequires regulatory approval, and this has not yet beengranted. Sensory evaluation panels are planned to testthese predictions. We also hope to determine theprecise olfactory or flavor roles of the other polysulfurvolatiles that have been produced.

Allium sulfur compounds are renowned for theirhuman health-giving attributes. We predict that thealtered profiles present in the reduced-LF plants arelikely to have significant consequences for these attri-butes; as such, they are being further investigated. Forexample, thiosulfinates have antiasthmatic activity(Griffiths et al., 2002). There is a clear rank order ofpharmacological activity, with saturated thiosulfinatesbeing less active than unsaturated ones. The unsaturated1-propenyl-containing thiosulfinate in the reduced-LFonions may confer health properties to onion thathave previously been associated with the unsaturatedallicin thiosulfinate in garlic. In addition, zwiebelaneshave been associated with anti-platelet-aggregationactivity (Block, 1992; Block et al., 1996b; Keusgen,2002). We are currently investigating the role of thezwiebelane isomer from our reduced-LF plants onplatelet aggregation. Benavides et al. (2007) demon-strated that di-2-propenyl disulfide from garlic isconverted after ingestion, by red blood cells, intohydrogen sulfide, a powerful signaling molecule thathas an ideal physiological profile for cardiovascularprotection (Lefer, 2007). The nature and similarity of

Figure 9. The proposed sulfur pathway following tissue disruption intransgenic onion. * Detected by pinking assay; ** detected by solventextraction GC-MS; *** detected by SPME-GC-MS.

Figure 10. The T-DNA region of the pART27-lfsRNAi-mgfp5ER silencing cassette used to silence LFS in onion. A 521-bp DNAfragment derived from lfs cDNA (AB089203) at positions 56 to 576 was selected for a sense lfs region of an RNAi construct andinserted into the XhoI and KpnI sites in pHANNIBAL. Similarly, a 512-bp DNA fragment at positions 63 to 574 was selected for anantisense lfs region and inserted into the ClaI and BamHI sites. The RNAi construct was excised from pHANNIBAL using therestriction enzyme NotI and inserted into the NotI site in the pART27-mgfp5ER. The gfp marker gene was used to aid the selectionof transgenic events. LB, Left border; RB, right border.





garlic disulfides to the disulfide compounds producedin the reduced-LF plants warrants investigation todetermine if they are also converted to hydrogensulfide in the blood.

In summary, these reduced-LF onions are a uniqueresource for understanding the role of specific sulfursecondary metabolites in plant biology, in humanhealth, and in terms of their potential value to theagrifood industry.

MATERIALS AND METHODS

Generation and Molecular Analysis ofTransgenic Plants

A mild hybrid mid-daylength fresh onion (Allium cepa ‘Enterprise’), a

pungent open-pollinated fresh onion (‘Pukekohe LongKeeper’), and a pun-

gent dehydration mid-daylength onion (Sensient Dehydrated Flavors) were

transformed, regenerated, and ex-flasked according to the method of Eady

et al. (2000). The T-DNA cassette designed for silencing in onion (Fig. 10) was

contained within a pArt binary vector (Gleave, 1992) with a m-gfpER reporter

gene under the control of a CaMV 35S promoter (Haseloff et al., 1997) and a

nptII gene under the control of a nos promoter for ease of detection and

selection. It contained the pHANNIBAL-based RNAi cassette (Wesley et al.,

2001) containing a 512-bp hairpin of the lfs gene sequence under CaMV 35S

promotional control. The CaMV35S-mgfpER and nos-nptII selection have

previously been shown to function well in onion (Eady et al., 2000). Southern-

blot analysis (Eady et al., 2000) gave integration details, loci, and copy

numbers. For subsequent leaf analysis, only healthy inner leaves (10–20 cm in

length) from actively growing plant material was used. To avoid any confu-

sion, only a single plant from each transgenic event, or a proven clone (by

Southern-blot analysis), was used for analysis. The presence of lfs siRNA

sequences was determined by the following simplified protocol of Hamilton

and Baulcombe (1999). Small RNA was isolated from approximately 450 mg of

leaf tissue using the mirVana miRNA isolation kit (Ambion) following the

manufacturer’s instructions. RNA was quantified using a GeneQuantII (GE

Healthcare Bioscience) and standardized to 250 ng mL21. Five micrograms in

40 mL (including loading buffer) was loaded onto a 15% polyacrylamide gel

and electrophoresed following the mirVana (Ambion) instructions. The separated

small RNA was capillary blotted onto a Zeta-probe GT membrane (Bio-Rad)

according to the manufacturer’s instructions, air-dried, and UV cross-linked

onto the membrane by a 120-mJ burst (BLX254; Vilber Lourmat). The blot was

prehybridized and incubated with a LFS probe, produced from a 450-bp LFS

sequence using a [32P]CTP random prime labeling kit (GE Healthcare Biosci-

ence) according to the manufacturer’s instructions, at 40�C overnight in a HB-

ID hybridizer (Techne). Excess probe was removed by two 30-min washes in

63 SSC, 0.2% SDS and then 23 SSC, 0.1% SDS. The washed membrane was

placed in a Lanex cassette against Bio-max film (Kodak) at 280�C for 2 weeks

before the film was developed.

For transcript analysis, total RNA was isolated from approximately 10 mg

of freeze-dried onion leaf tissue using an RNeasy Plant Mini Kit (QIAGEN)

following the manufacturer’s instructions. RNA was quantified using a ND-

1000 spectrophotometer (Nano Drop Technologies) after treatment with

TURBO DNA-free (Ambion). Two hundred nanograms of the total RNA was

reverse transcribed to first-strand cDNA using the Omniscript RT Kit

(QIAGEN) and oligo(dT)21 primer.

For qualitative PCR to confirm the presence of lfsRNAi transcripts, the

border region between antisense lfs of the lfsRNAi cassette and the ocs

terminator was selected as a target to be amplified using specific primers

(forward, 5#-CTCTTCGATTTTCTGACCTATCTCAGTAGC-3#; reverse, 5#-TGC-

ACAACAGAATTGAAAGC-3#). The target sequence was amplified in a

25-mL reaction volume containing the first-strand cDNA, equivalent to 10 ng

of total RNA, using a HotStarTaq Master Mix Kit (QIAGEN) according to the

manufacturer’s instructions. PCR, performed in a GeneAmp 9700 (Applied

Biosystems), was set with the following PCR cycle program: 15 min at 95�C

and 40 cycles of 1 min at 94�C, 1 min at 55�C, 1 min at 72�C, and 10 min at 72�C.

For quantitative real-time PCR, the 5# region of lfs (accession no. AB089203)

was selected as a target to be amplified using specific primers (forward,

5#-ACGTTATATCAAGAAGATTGTCCAA-3#; reverse, 5#-TCCGTTAGCAC-

TATCAGCGAC-3#) and putative ubiquitin sequence (accession no. AA451588)

was selected as an internal control gene to be amplified using specific primers

(forward, 5#-ACGATTACACTAGAGGTGGAGAGCTC-3#; reverse, 5#-CCT-

GCAAATATCAGCCTCTGCT-3#). Each sequence was amplified in a 25-mL

reaction volume containing the first-strand cDNA, equivalent to 10 ng of total

RNA, using the QuantiTect SYBR Green PCR Kit (QIAGEN) according to the

manufacturer’s instructions. PCR, performed in an ABI PRISM 7700 (Applied

Biosystems), was set with the following PCR cycle program: 15 min at 95�C

and 40 cycles of 15 s at 94�C, 30 s at 57�C, and 30 s at 72�C. Each standard curve

was calibrated using five concentrations of RT-PCR amplicon containing lfs or

putative ubiquitin sequence, such as 100, 1,000, 10,000, 100,000, and 1,000,000

copies per reaction. The lfs mRNA level was determined by dividing the lfs copy

number by the internal standard gene copy number as a relative mRNA level.

Biochemical Analysis of Plants

Western Analysis of LFS Protein

Total protein was extracted from approximately 20 mg of freeze-dried

onion leaf tissue by PBS buffer and quantified using a protein assay (Bio-Rad)

with bovine serum albumin (Sigma) as a standard. One hundred micrograms

of protein in Laemmli Sample Buffer (Bio-Rad) was loaded onto a 15%

polyacrylamide gel and electrophoresed. The protein was electrically blotted

using a Trans-Blot SD Cell (Bio-Rad) onto a polyvinylidene difluoride mem-

brane (Bio-Rad) following the manufacturer’s instructions. The blot was

blocked by BSA solution and incubated for 1 h with an LFS polyclonal

antibody raised in a rabbit. Excess antibody was removed by two 20-min

washes in PBS buffer, and the membrane was incubated for 1 h with an anti-

rabbit IgG solution. Again, excess antibody was removed by three 20-min

washes in PBS buffer, and the LFS protein was detected using the HRP

Conjugate Substrate Kit (Bio-Rad) according to the manufacturer’s instruc-

tions. The detected membranes were incorporated by ChemiDoc XRS (Bio-

Rad) according to the manufacturer’s instructions. Both LFS and degraded

LFS bands were selected and quantified using Quantity One Software version

4.6 (Bio-Rad) according to the manufacturer’s instructions.

LF Analysis

Approximately 2 g of young leaf from the second or third innermost leaf of

actively growing glasshouse plants and mature bulb samples (top half

bisected longitudinally) was harvested into liquid N2. Samples were stored

at 280�C until ready for analysis. Half of each leaf and bulb sample was freeze

dried, stored at room temperature in a desiccator, and used for LF assays and

precursor evaluation (in vivo LF leaf assays used fresh frozen material).

In Vivo LF Assays

Sample preparation and GC analysis for LF were modified from a previ-

ously reported method (McCallum et al., 2005). Samples of leaf (20–100 mg)

were crushed rapidly in 500 mL of deionized water in 1.5-mL microcentrifuge

tubes. The tubes were held at room temperature (approximately 20�C) with

stirring every minute. Three minutes after the initial mixing, 500 mL of

dichloromethane (containing 0.06 mL mL21 heptane thiol as an internal

standard) was added. The tubes were gently rolled for 2 min to extract the

sulfur compounds. A 300-mL sample of the dichloromethane phase was

removed to a GC autoinjector vial for immediate analysis.

Samples of bulb powder (10–30 mg) were prepared as above, except that in

addition to 500 mL of deionized water, seven times the sample weight of water

was added to the sample to replace the water removed by freeze drying.

GC operating conditions were modified from the previous method as

follows: 1-mL splitless injection with injector at 160�C; hydrogen carrier gas at

constant 60 cm s21 flow; isothermal 55�C oven temperature; flame photometric

detector at 180�C. The detector was calibrated against heptane thiol. Three

samples were prepared for analysis from each leaf and bulb powder sample,

and duplicate GC runs were done for each of the replicate samples. Mean

(n 5 6) LF levels are expressed as picograms of LF per gram fresh weight using

the dry weights of the bulb samples to adjust to a fresh weight basis.

In Vitro LF Assays

LFS enzyme activity was evaluated by LF-forming capability. Samples of

freeze-dried powder (5–15 mg) were mixed with PBS buffer (137 mM NaCl, 8.1

Eady et al.




mM Na2HPO4, 2.68 mM KCl, and 1.47 mM KH2PO4) at a concentration of 1 g per

100 mL. The mixture was centrifuged for 5 min at 15,000 rpm at 4�C, and the

resulting supernatant was used for the activity assay. Protein quantification

was performed as above. For the LF-forming capability assay, 10 mL of the

extracted protein solution, 40 g mL21 purified garlic (Allium sativum) alliinase

(555 nkat mg21 protein), and 20 g mL21 of 20 mg mL21 trans-PRENCSO

purified from onion were mixed and left for 3 min at room temperature. Two

microliters of the resulting mixture was analyzed by HPLC using an ODS

column at 35�C. The column was eluted with 30% (v/v) acidic methanol (pH

3.3) at a flow rate of 0.6 mL min21, and LF was monitored by the A254. LFS

enzyme activity was assessed by calculating LF peak area per nanogram of

protein in 2 mL of the reaction mixture.

PRENCSO Substrate and Alliinase Activity

PRENCSO precursor levels were assayed as thiocarbamate derivatives by

reverse-phase HPLC following the protocol of Randle et al. (1995) Alliinase

activity levels were determined following a modified Schwimmer and Mazelis

method (Lancaster et al., 2000) further adapted by the use of propyl-Cys

sulfoxide as the substrate and a higher, 30�C reaction temperature.

Pinking Assay

The range and reliability of the pinking assay were examined. Several

volumes of 20 mg mL21 1-PRENCSO or alliin (0–850 nmol) were mixed with

40 mL of 5% Gly solution and 145 mL (20 nkat) of purified garlic alliinase (555

nkat mg21 protein). After 30 min at 25�C, insoluble matter was removed by

centrifugation and 10 mL of 100 mL L21 formaldehyde solution was added to

100 mL of the supernatant. This was then incubated at 40�C for 6 h, and then

A520 was measured by spectrophotometry. Each assay was performed in

triplicate.

The pinking assay was developed as follows: 30 mg of freeze-dried bulb

powder was resuspended in 200 mL of 1% Gly solution with and without 15

mg of recombinant LFS. After 30 min at 25�C, insoluble matter was removed by

centrifugation and 10 mL of 100 mL L21 formaldehyde solution was added to

100 mL of the supernatant. This was incubated at 40�C for 6 h, and then A520

was measured by spectrophotometry. Increasing absorbance (pinking) dem-

onstrated the relative abundance of 1-propenyl-containing thiosulfinates.

Head Space SPME-GC-MS Analysis

Head space analysis was performed according to Hori’s method (2007)

with slight modifications. Five grams of sliced fresh leaf material from each

plant was put into 20-mL SPME vials and covered with a lid immediately, then

left for 30 min at room temperature. The head space of the vial was extracted

with SPME (100 mM polydimethylsiloxane; Supelco) at room temperature for

10 min. The fiber was inserted into a 5975C inert XL MSD MS system (Agilent

Technologies) equipped with a 7890A GC system (Agilent Technologies). Data

processing used MSD ChemStation Data Analysis (Agilent Technologies). GC

separation was achieved using a 30-m 3 0.25-mm i.d. DB-5ms column with

0.25-mm film (Agilent Technologies). The carrier was 99.999% helium at a

column pressure of 100 kPa, and the column temperature program was 5 min

at 35�C, 4�C min21 to 200�C, 16 min at 200�C, and 10�C min21 to 250�C.

Transfer line and detector temperature was held at 150�C, and total ion

chromatograms and mass spectra were analyzed with the electron-impact

mode. The compounds were identified by matching their mass spectra to the

U.S. National Institute of Standards and Technology and Wiley mass spectral

libraries and previous studies (Hiramitsu, 1989; Sinha et al., 1992; Block et al.,

1996a).

Solvent Extraction GC-MS Analysis

GC-MS analysis was performed according to Arnault’s method (2000) with

slight modifications. Forty milligrams of freeze-dried bulb powder from each

plant was incubated in 400 mL of deionized water at room temperature and

vortexed gently. After 80 min, 200 mL of diethyl ether (containing 0.01 mL of

benzyl alcohol as an internal standard) was added and vortexed briefly. After

1 min of centrifugation at 15,000 rpm, the diethyl ether phase was transferred

to a GC autoinjector vial for immediate analysis. One microliter was injected

into a 5975C inert XL MSD MS system (Agilent Technologies) equipped with a

7890A GC system (Agilent Technologies). Data processing used MSD Chem-

Station Data Analysis (Agilent Technologies). GC separation was achieved

using a 15-m 3 0.32-mm i.d. DB-1 column with a thick coating (5 mm; Agilent

Technologies). The carrier was 99.999% helium at 3.5 mL min21, and the

column temperature program was 5�C min21 from 70�C to 250�C. Transfer line

and detector temperature was held at 150�C, and total ion chromatograms and

mass spectra were analyzed with the electron-impact mode. The compounds

were identified by matching their mass spectra to the U.S. National Institute of

Standards and Technology and Wiley mass spectral libraries and previous

studies (Block et al., 1992b; Arnault et al., 2000).

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession number AB089203.

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure S1. Mass spectral data.

Supplemental Figure S2. Proposed reaction mechanism for producing a

pink pigment.

ACKNOWLEDGMENTS

We thank Peter Waterhouse of the Commonwealth Scientific and Indus-

trial Research Organization for the use of RNAi technology and critical

review of the manuscript. Within Crop and Food Research, we thank Ruth

Butler for statistical analysis, Doug Taylor for plant maintenance, and Daniel

Park for editing.

Received May 22, 2008; accepted June 10, 2008; published June 26, 2008.

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